Apigenin Regulates Interleukin-1Β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

Original Article Biomol Ther 24(2), 163-170 (2016)

Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

Jin Sung Park1,†, Dong Kyu Kim2,†, Hyun-Dae Shin2, Hyun Jae Lee3, Ho Seung Jo1, Jin Hoon Jeong1, Young Lac Choi1, Choong Jae Lee3,* and Sun-Chul Hwang1,* 1Department of Orthopedic Surgery and Institute of Health Sciences, School of Medicine and Hospital, Gyeongsang National University, Jinju 52727, 2Department of Orthopedic Surgery, School of Medicine, Chungnam National University, Daejeon 35015, 3Department of Pharmacology, School of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea

Abstract We examined whether apigenin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effects of apigenin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription - polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of apigenin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of apigenin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, apigenin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Fur- thermore, apigenin inhibited the secretion and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that apigenin can regulate the gene expression, secretion, and activity of MMP-3, by directly acting on articular chondrocytes.

Key Words: Apigenin, Chondrocyte, Metalloproteinase, Osteoarthritis

INTRODUCTION lar cartilage, the matrix metalloproteinases (MMP) play a pivotal role in the destruction of articular cartilage in osteoarthritis Osteoarthritis has been reported to be the most common patients (Dean et al., 1989; Kullich et al., 2007). MMPs can be degenerative articular disease, from which millions of people classified into collagenases (MMP-1, -8 and -13), gelatinases suffered particularly in the elderly. Among the symptoms of (MMP-2 and -9), and stromelysins (MMP-3, -7, -10 and -11) osteoarthritis, degeneration of articular cartilage, formation (Birkedal-Hansen et al., 1993; Burrage et al., 2006). Out of of osteophytes, synovial inflammation, and changes in sub- these MMPs, MMP-3 degrades proteoglycans and activates chondral bone are the major pathophysiologic features. Al- procollagenase in articular cartilage (Garnero et al., 2000; Lin though the cause of osteoarthritis is not fully understood, it et al., 2004). Since some plant-derived natural products used involves several biochemical and mechanical factors, such as as arthritis remedies in folk medicine, exploration of the in- disruption on the equilibrium between physiologic synthesis hibition mechanisms of the proteolytic activity, secretion, and and degradation of articular cartilage during the progression gene expression of MMP-3 by these plant-derived products of osteoarthritis (Mankin, 1982; Aigner and McKenna, 2002). will support the effective treatment of osteoarthritis, and may While the activation of degradative enzymes leads to the expedite a new therapeutic strategies. loss and degradation of proteoglycans and collagen in articu- As claimed by various reports, apigenin derived from Scu-

Open Access http://dx.doi.org/10.4062/biomolther.2015.217 Received Dec 30, 2015 Revised Jan 17, 2016 Accepted Jan 22, 2016 Published Online Mar 1, 2016 This is an Open Access article distributed under the terms of the Creative Com- mons Attribution Non-Commercial License (http://creativecommons.org/licens- *Corresponding Authors es/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] (Lee CJ), [email protected] (Hwang SC) and reproduction in any medium, provided the original work is properly cited. Tel: +82-42-580-8255 (Lee CJ), +82-55-750-8102 (Hwang SC) Fax: +82-42-585-6627 (Lee CJ), +82-55-750-8104 (Hwang SC) †The first two authors contributed equally to this work. Copyright © 2016 The Korean Society of Applied Pharmacology www.biomolther.org

163 Biomol Ther 24(2), 163-170 (2016)

tellariae Radix, a medicinal plant used for controlling the in- cells were incubated for 2 h in growth medium with 1, 10, 50, flammatory diseases in folk medicine, showed the various or 100 μM of apigenin, hesperidin or hesperetin, followed by biological activities including anti-inflammatory and anti-MMP incubation in the presence or absence of IL-1β (10 ng/mL) for effects (Durigova et al., 2008; Hwang et al., 2011; Palmieri 24 h. Apigenin, hesperidin or hesperetin was dissolved in di- et al., 2012; Du et al., 2015; Patil et al., 2015). For instance, methylsulfoxide, diluted in PBS, and administered in culture apigenin showed the inhibitory activities on MMP-9 in rats with medium (final concentrations of dimethylsulfoxide were 0.5%). acute myocardial infarction (Du et al., 2015) and in endothe- The final pH values of these solutions were between 7.0 and lial cells (Palmieri et al, 2012). Also, apigenin inhibited both 7.4. Culture medium and 0.5% dimethylsulfoxide in medium MMP-1 activity in human keratinocytes (Hwang et al., 2011) did not affect the gene expression, secretion, or proteolytic and hyaluronidase activity in cartilage (Durigova et al., 2008). activity of MMP-3 in primary cultured chondrocytes. The su- However, there are no research result about the effect of pernatant was collected and centrifuged, and cell and super- apigenin on the gene expression, secretion, and activity of natant fractions were stored at -80°C until use. MMP-3, an articular cartilage-degradative enzyme that decomposes proteoglycans, especially in primary cultured rabbit Cytotoxicity assay articular chondrocytes, or on in vivo production of MMP-3 in Chondrocytes were seeded at a density of 2×105/mL (0.1 the rat knee. Thus, to evaluate the chondroprotective potential mL/well) in a 96-well microtiter plate, and allowed to attach for of apigenin, we investigated its effects on IL-1β-induced gene 24 h to keep the log phase growth at the time of drug treat- expression, secretion, and activity of MMP-3 in vitro, and on ment. Apigenin was dissolved in DMSO, and administered in production of MMP-3 in vivo. DMEM supplemented with 10% FBS (final concentrations of DMSO were under 0.5%). 0.5% DMSO alone did not affect the proliferation of chondrocytes. After incubation with the MATERIALS AND METHODS indicated drug concentrations for 72 h, cell proliferation was determined using the sulforhodamine B (SRB) assay (Skehan Materials et al., 1990). All chemicals and reagents used in this experiment, including apigenin (purity: 98.0%), hesperidin (purity: 98.0%) and Isolation of total RNA and RT-PCR hesperetin (purity: 98.0%) were purchased from Sigma-Al- Total RNA was isolated from chondrocytes using the Easy- drich (St. Louis, MO, USA) unless otherwise specified. Dul- BLUE Extraction Kit (INTRON Biotechnology, Inc., Kyung- becco’s Modified Eagle’s Medium (DMEM) was purchased ki-do, Korea), and reverse transcribed using AccuPower RT from Gibco-BRL (Grand Island, NY, USA) and recombinant Premix (BIONEER Corporation, Daejeon, Korea) according to human IL-1β was purchased from R&D Systems (Minneapo- the manufacturer's instructions. About 2 mg of total RNA was lis, MN, USA). primed with 1 mg of oligo (dT) in a final reaction volume of 30 mL. 2 mL of RT reaction product was amplified in 20 mL using Primary cultures of chondrocytes from rabbit articular Thermoprime Plus DNA Polymerase (ABgene, Rochester, NY, cartilage USA). PCR was performed with the following primers: MMP- Male New Zealand White rabbits were obtained from Dae- 3 (5′ATG GAC CTT CTT CAG CAA 3′, 5′TCA TTA TGT CAG han Biolink (Seoul, Korea) at 2 weeks of age. Animals were CCT CTC 3′), MMP-13 (5′AGG AGC ATG GCG ACT TCT AC housed 1 animal per cage, provided with distilled water and 3′, 5′TAA AAA CAG CTC CGC ATC AA 3′), MMP-1 (5′TCA food ad libitum, and kept under a 12 h light/dark cycle (lights GTT CGT CCT CAC TCC AG 3′, 5′TTG GTC CAC CTG TCA on from 08:00-20:00) at constant temperature (22.5°C) and TCT TC 3′), a disintegrin and metalloproteinase with thrombo- humidity (55%). Animals were cared for in accordance with spondin motifs-4 (ADAMTS-4) (5′CAA GGT CCC ATG TGC the Guide for the Care and Use of Laboratory Animals, and AAC GT 3′, 5′CAT CTG CCA CCA CCA GTG TCT 3′), and AD- care was regulated by Chungnam National University (the ap- AMTS-5 (5′TGT CCT GCCAGC GGATGT 3′; 5′ACG GAA TTA proval number of animal experiment: CNU-00555) (Daejeon, CTG TAC GGC CTA CA 3′). GAPDH (5′ACT GGC GTC TTC Korea). Rabbit articular chondrocytes were isolated from the ACC ACC AT 3′; 5′AAG GCC ATG CCA GTG AGC TT 3′) was tibial plateau and femoral condyle in cartilage of the knee joint. used as a quantitative control. The PCR products increased Cartilage was washed in phosphate-buffered saline (PBS) and as the concentration of RNA increased. The amplified frag- minced into pieces measuring 2 mm3, approximately. Carti- ment sizes were 350 base pairs (bp) for MMP-3, 458 bp for lage tissue was digested for 4 h with 0.2% type II collagenase MMP-13, 300 bp for MMP-1, 90 bp for ADAMTS-4, 110 bp for at 37°C. After collection of individual cells by brief centrifu- ADAMTS-5, and 400 bp for GAPDH. After PCR, 15 mL of PCR gation, the cells were transferred to 100 mm culture dishes products were subjected to 2% agarose gel electrophoresis (seeding density: 105 cells/cm2) in 12 mL DMEM supplement- and visualized with ethidium bromide under a transilluminator. ed with 10% fetal bovine serum (FBS), in the presence of The signal intensity of each band was analyzed by GelQuant penicillin (100 units/mL) and streptomycin (100 μg/mL) (Moon software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel) et al., 2011). Cells were cultured at 37°C in a humidified, 5% (Moon et al., 2011). CO2/95% air, water-jacketed incubator, and medium was re- placed every other day. Western blot analysis for measuring secretion level of MMP-3 in culture supernatant Treatment of cells with apigenin and the other natural The Bradford assay was used to measure protein concen- products trations in culture supernatants to ensure consistent weight of Chondrocytes were seeded on 6-well culture plates at a protein samples subjected to electrophoresis. Culture super- density of 105 cells/cm2. After 2 days in monolayer culture, the natant samples containing MMP-3 protein (50 mg each) were http://dx.doi.org/10.4062/biomolther.2015.217 164 Park et al. Apigenin and Osteoarthritis

A B b b b b b b b 1 1 b 1 1 1

Cont IL-1b Hesperidin1+IL-1Hesperidin10+IL-Hesperidin50+IL-Hesperidin100+IL-1 Cont IL-1b Hesperetin1+IL-Hesperetin10+IL-Hesperetin50+IL-Hesperetin100+IL-1 MMP-3 MMP-3

GAPDH GAPDH Hesperidin-MMP-3 gene expression Hesperetin-MMP-3 gene expression 300 500 250

control) * control) 400 * (% 200 (% 300 150 200

intensity 100 intensity 50 100 0 0 Relative Cont IL-1b 11050 100 Relative Cont IL-1b 11050 100 Treatment Treatment C b b b 1 b 1 1 1

Cont IL-1b Apigenin1+IL-Apigenin10+IL-Apigenin50+IL-Apigenin100+IL- MMP-3

GAPDH Apigenin-MMP-3 gene expression 1,000

control) 800

(% 600 *

400

intensity 200

0 Relative Cont IL-1b 11050 100 Treatment

Fig. 1. Effect of hesperidin, hesperetin or apigenin on MMP-3 gene expression in rabbit chondrocytes. Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 mM) of hesperidin, hesperetin or apigenin for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. MMP-3 gene expression level was measured by RT-PCR. Three independent experiments were performed and the representative data were shown (A, B and C). The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents a mean ± S.E.M. of three independent experiments in comparison with that of the control set at 100%. *Significantly different from control (p<0.05). †Significantly different from IL-1β alone (p<0.05). cont: control, concentration unit is mM.

subjected to 10% sodium dodecyl sulfate-polyacrylamide miluminescence kit (Pierce ECL western blotting substrate, gel electrophoresis (SDS-PAGE), and then transferred onto Thermo Scientific, Waltham, MA, USA). The signal intensity a polyvinylidene difluoride (PVDF) membrane. Blots were of immunoreactive bands was analyzed by GelQuant software blocked using 5% skim milk in Tris-buffered saline/Tween 20 (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). (TBS-T), and probed overnight with MMP-3 antibody in blocking buffer at 4°C. Antibody against MMP-3 was purchased Casein zymography to measure the proteolytic activity of from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mem- MMP-3 branes were washed with TBS-T and probed for 1 h with a A modified casein-substrate zymography was carried out secondary antibody conjugated with horseradish peroxidase using culture supernatants from chondrocytes pretreated for (Calbiochem, La Jolla, CA, USA). After 4 washes with TBS-T, 2 h with apigenin and stimulated for 24 h with IL-1β in DMEM immunoreactive bands were detected using an enhanced che- containing 0.5% FBS. The Bradford assay was used to mea-

165 www.biomolther.org Biomol Ther 24(2), 163-170 (2016)

150 sure protein concentrations in culture media to ensure consis- tency across samples. Samples were electrophoresed at 4°C

control) in a 10% SDS gel containing 0.1% casein. After electrophore-

(% 100 sis, gels were washed with 10 mM Tris-HCl (pH 8.0) containing 2.5% Triton X-100. Next, gels were incubated at 37°C for 48 h in 50 mM Tris-HCl (pH 8.0) containing 1% Triton X-100, 50 0.2 M NaCl, and 5 mM CaCl2. Finally, gels were stained with

proliferation 1% Coomassie Brilliant Blue, destained, and photographs were taken. The signal intensity of each band was analyzed Cell 0 Cont 11050 100 by GelQuant software (DNR Bio-Imaging Systems Ltd., Jeru- salem, Israel) (Moon et al., 2011). Treatment (Mm ) Fig. 2. Effect of apigenin on proliferation of rabbit chondrocytes. In vivo experiments Chondrocytes were incubated for 72 h in the presence of vary- Male Sprague-Dawley rats (Daehan Biolink, Seoul, Korea) ing concentrations of apigenin. Cell viability was determined us- weighing 200-210 g were used to investigate the effect of api- ing SRB assay as described in Materials and Methods. Each bar genin on production of MMP-3 in articular cartilage in vivo. represents a mean ± S.E.M. of three independent experiments in Animals were housed 5 per cage, provided with distilled water comparison with that of the control set at 100%. and food ad libitum, and kept under a 12 h light/dark cycle (lights on from 08:00-20:00) at constant temperature (22.5°C) and humidity (55%). Animals were cared for in accordance RESULTS with the Guide for the Care and Use of Laboratory Animals, and care was regulated by Chungnam National University Effect of apigenin, hesperidin or hesperetin on MMP-3 (the approval number of animal experiment: CNU-00555) gene expression in rabbit chondrocytes (Daejeon, Korea). Rats were randomly divided into 4 groups: To compare the potency of activity on the gene expression control, IL-1β only, 50 μM apigenin plus IL-1β, or 100 μM api- of MMP-3, the key MMP involved in destruction of articular genin plus IL-1β. Rats were anesthetized with vaporized di- cartilage, effect on MMP-3 gene expression was investigated ethyl ether, and those from the 50 μM apigenin plus IL-1β and by pretreatment of hesperidin or hesperetin-similar flavonoidal 100 μM apigenin plus IL-1β treatment groups received a 30 μL compounds with apigenin-in addition to apigenin. As shown in injection of 50 μM or 100 μM apigenin, respectively, into the Fig. 1C, apigenin inhibited IL-1β-induced MMP-3 gene expres- right knee joint. After 3 h, rats from the IL-1β only group, the 50 sion. However, hesperidin or hesperetin did not affect MMP-3 μM apigenin plus IL-1β group, and the 100 μM apigenin plus gene expression (Fig. 1A, 1B). The relative signal intensity of IL-1β group received a 30 μL injection of 20 ng IL-1β in sterile each band of apigenin-treated cultures were 100 ± 36%, 516 ± PBS into the right knee joint. Rats from the control group were 23%, 506 ± 17%, 486 ± 27%, 238 ± 13% and 196 ± 27% for injected with 30 μL of sterile PBS. Rats were euthanized via control, 10 ng/mL of IL-1β alone, IL-1β plus apigenin 1 μM, IL- CO2 asphyxiation 72 h after injections. Articular cartilage (tibial 1β plus apigenin 10 μM, IL-1β plus apigenin 50 μM, and IL-1β plateau and femoral condyle) was isolated from each animal, plus apigenin 100 μM, respectively. The relative signal inten- homogenized, and prepared for measurement of MMP-3 pro- sity of each band of hesperidin-treated cultures were 100 ± tein by western blot analysis. Tissue lysates from articular car- 10%, 209 ± 21%, 192 ± 17%, 185 ± 23%, 192 ± 14% and 187 ± tilage homogenates containing MMP-3 protein (50 mg each) 19% for control, 10 ng/mL of IL-1β alone, IL-1β plus hesperi- were subjected to 10% SDS-PAGE, and transferred onto a din 1 μM, IL-1β plus hesperidin 10 μM, IL-1β plus hesperidin PVDF membrane. Blots were blocked with 5% skim milk in 50 μM, and IL-1β plus hesperidin 100 μM, respectively. The TBS-T, and probed with MMP-3 antibody (Santa Cruz Biotech- relative signal intensity of each band of hesperetin-treated cul- nology, Santa Cruz, CA, USA) in blocking buffer overnight at tures were 100 ± 21%, 309 ± 17%, 287 ± 26%, 316 ± 21%, 4ºC. Membranes were washed with TBS-T, and probed for 1 327 ± 13% and 306 ± 29% for control, 10 ng/mL of IL-1β alone, h with a secondary antibody conjugated with horseradish per- IL-1β plus hesperetin 1 μM, IL-1β plus hesperetin 10 μM, IL- oxidase (Calbiochem, La Jolla, CA, USA). After 4 washes with 1β plus hesperetin 50 μM, and IL-1β plus hesperetin 100 μM, TBS-T, immunoreactive bands were detected using an en- respectively. hanced chemiluminescence kit (Pierce ECL western blotting substrate, Thermo Scientific, Waltham, MA, USA). The signal Effect of apigenin on proliferation of rabbit chondrocytes intensity of immunoreactive bands was analyzed by GelQuant (cytotoxicity assay) software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). To investigate the potential cytotoxicity of apigenin to cultured rabbit chondrocytes, effect of apigenin on proliferation of Statistics rabbit chondrocytes using SRB assay was tested. As can be Means of individual group were converted to percent con- seen in Fig. 2, apigenin showed no significant cytotoxicity at trol and expressed as mean ± S.E.M. The difference between the concentrations of 1, 10, 50, and 100 mM. The numbers of groups was assessed using one-way ANOVA and Holm-Sidak cells in apigenin-treated cultures were 100 ± 20%, 90 ± 16%, test as a post-hoc test. p<0.05 was considered as significantly 112 ± 9%, 86 ± 21%, and 92 ± 18% for control, 1, 10, 50, and different. 100 μM apigenin, respectively.

Effect of apigenin on the gene expression of MMP-1, MMP- 13, ADAMTS-4, and ADAMTS-5 in rabbit chondrocytes If apigenin can affect the gene expression of MMP-3, the http://dx.doi.org/10.4062/biomolther.2015.217 166 Park et al. Apigenin and Osteoarthritis

b b b b 1 b b 1 b 1 b 1 1 1

Cont IL-1b Apigenin1+IL-1Apigenin10+IL-Apigenin50+IL-Apigenin100+IL- Cont IL-1b Apigenin1+IL-Apigenin10+IL-1Apigenin50+IL-Apigenin100+IL- MMP-1 MMP-3 Secretion

MMP-13 b-actin MMP-3 Casein zymography ADAMTS-4 ADAMTS-5 MMP-3 secretion&activity GAPDH 1,400 Secretion Gene expression 1,200 Activity 800 MMP-1 control) * 1,000 MMP-13 (% control) 600 ADAMTS-4 800 * (% ADAMTS-5 * 600 * 400 intensity 400 * 200

intensity * 200 0 Relative Cont IL-1b 11050 100 0 Relative Treatment (Mm ) Cont IL-1b 11050 100 Treatment (Mm ) Fig. 4. Effect of apigenin on IL-1β-induced secretion of MMP-3 and caseinolytic activity of MMP-3 in rabbit articular chondrocytes. Fig. 3. Effect of apigenin on the gene expression of MMP-1, Primary cultured rabbit articular chondrocytes were pretreated MMP-13, ADAMTS-4, or ADAMTS-5 in rabbit chondrocytes. Pri- with varying concentrations (1, 10, 50, and 100 mM) of apigenin mary cultured rabbit articular chondrocytes were pretreated with for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. Culture varying concentrations (1, 10, 50, and 100 mM) of apigenin for 2 supernatants were collected for measurement of both the levels of h and then stimulated with IL-1β (10 ng/mL) for 24 h. The gene produced and secreted MMP-3 by western blot analysis and the expression level of MMP-1, MMP-13, ADAMTS-4, or ADAMTS-5 proteolytic activity of MMP-3 by casein zymography. The fourth was measured by RT-PCR. Three independent experiments were panel was black-and-white version of casein zymography. Three performed and the representative data were shown. The signal independent experiments were performed and the representative intensity of each band was analyzed by GelQuant software (DNR data were shown. The signal intensity of each band was analyzed Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, a mean ± S.E.M. of three independent experiments in comparison Israel). Each bar represents a mean ± S.E.M. of three independent with that of the control set at 100%. *Significantly different from experiments in comparison with that of the control set at 100%. control (p<0.05). †Significantly different from IL-1β alone (p<0.05). *Significantly different from control (p<0.05). †Significantly different Cont: control, concentration unit is mM. from IL-1β alone (p<0.05). Cont: control, concentration unit is mM.

key MMP involved in destruction of articular cartilage, it should plus apigenin 100 μM, respectively. The relative signal intensi- be investigated whether apigenin affect the gene expression of ty of each band of apigenin-treated cultures for measuring the MMP-1, MMP-13, ADAMTS-4 or ADAMTS-5, the other degra- effect on ADAMTS-5 were 100 ± 7%, 235 ± 12%, 206 ± 8%, dative enzymes related to destruction of articular cartilage, in 150 ± 10%, 132 ± 12% and 106 ± 9% for control, 10 ng/mL of rabbit chondrocytes. As shown in Fig. 3, apigenin showed the IL-1β alone, IL-1β plus apigenin 1 μM, IL-1β plus apigenin 10 inhibition of IL-1β-induced gene expression of MMP-1, MMP- μM, IL-1β plus apigenin 50 μM, and IL-1β plus apigenin 100 13, ADAMTS-4, and ADAMTS-5. The relative signal intensity μM, respectively. of each band of apigenin-treated cultures for measuring the effect on MMP-1 were 100 ± 17%, 418 ± 21%, 236 ± 15%, Effect of apigenin on IL-1β-induced secretion of MMP-3 221 ± 13%, 196 ± 12% and 113 ± 9% for control, 10 ng/mL of from rabbit articular chondrocytes IL-1β alone, IL-1β plus apigenin 1 μM, IL-1β plus apigenin 10 If apigenin can affect the MMP-3 gene expression at the μM, IL-1β plus apigenin 50 μM, and IL-1β plus apigenin 100 transcriptional level, it can be examined whether apigenin af- μM, respectively. The relative signal intensity of each band of fects IL-1β-induced secretion of MMP-3 proteins from rabbit apigenin-treated cultures for measuring the effect on MMP-13 articular chondrocytes. As shown in Fig. 4, stimulation with IL- were 100 ± 9%, 283 ± 16%, 226 ± 12%, 236 ± 18%, 215 ± 16% 1β (10 ng/mL) increased the secretion of MMP-3 from chon- and 198 ± 13% for control, 10 ng/mL of IL-1β alone, IL-1β plus drocytes. However, apigenin inhibited the effects of IL-1β on apigenin 1 μM, IL-1β plus apigenin 10 μM, IL-1β plus apigenin MMP-3 secretion. This result means that apigenin can control 50 μM, and IL-1β plus apigenin 100 μM, respectively. The rela- the steps of protein synthesis and secretion of MMP-3, maybe tive signal intensity of each band of apigenin-treated cultures due to a result of reduced MMP-3 mRNA level. The relative for measuring the effect on ADAMTS-4 were 100 ± 21%, 506 signal intensity of each immunoreactive band of apigenin- ± 28%, 426 ± 36%, 390 ± 21%, 226 ± 18% and 118 ± 12% for treated cultures were 100 ± 26%, 978 ± 38%, 816 ± 29%, 687 ± control, 10 ng/mL of IL-1β alone, IL-1β plus apigenin 1 μM, IL- 42%, 564 ± 56% and 406 ± 31% for control, 10 ng/mL of IL-1β 1β plus apigenin 10 μM, IL-1β plus apigenin 50 μM, and IL-1β alone, IL-1β plus apigenin 1 μM, IL-1β plus apigenin 10 μM,

167 www.biomolther.org Biomol Ther 24(2), 163-170 (2016)

b b 1 intensity of each immunoreactive band of apigenin-treated cultures were 100 ± 9%, 190 ± 10%, 147 ± 7% and 116 ± 8% for control, 20 ng/30 μL of IL-1β alone, IL-1β plus apigenin 50 μM, and IL-1β plus apigenin 100 μM, respectively. Cont IL-1b Apigenin50+IL-Apigenin100+IL-1 MMP-3

b-actin DISCUSSION In vivo MMP-3 production 400 As an effort to control the disrupted equilibrium between synthesis and degradation of articular cartilage during the

control) 300 progression of osteoarthritis, development of a useful phar-

(% macological tool can be a promising approach. Among well- 200 * known and commonly used arthritis remedies in folk medicine, apigenin is a medicinal plant-derived flavonoid that has shown intensity 100 anti-inflammatory and anti-matrix metalloproteinase effects (Durigova et al., 2008; Hwang et al., 2011; Palmieri et al., 0 2012; Du et al., 2015; Patil et al., 2015). However, no informa- Relative Cont IL-1b 50 100 tion has been reported on the effects of apigenin on the gene Treatment expression, secretion, and enzyme activity of MMP-3, which is an articular cartilage-degradative enzyme that decomposes Fig. 5. Effect of apigenin on the production of MMP-3 in vivo. The proteoglycans. Therefore, we examined the effects of apigenin m knee joint of rats were pretreated with 50 or 100 M of apigenin on MMP-3 gene expression, secretion, and proteolytic activity for 3 h and then stimulated with IL-1β (20 ng/30 μL) for 72 h, by intraarticular injection. Tissue lysates from articular cartilage homog- in primary cultured rabbit articular chondrocytes, as well as on enates containing MMP-3 proteins were collected for measurement MMP-3 production in the rat knee. of the level of produced MMP-3 in vivo, by western blot analysis. Even as defined as a non-inflammatory disease, develop- The representative data were shown. Equal protein loading was ment and progression of osteoarthritis have been caused by evaluated by β-actin levels. The signal intensity of immunoreactive low-grade inflammation in intraarticular sites, as well as to bands was analyzed by GelQuant software (DNR Bio-Imaging Sys- various inflammatory cytokines in articular tissues and fluids tems Ltd., Jerusalem, Israel). Each bar represents a mean ± S.E.M. of three independent experiments in comparison with that of the that are produced by chondrocytes and/or interact with chon- control set at 100%. *Significantly different from control p( <0.05). drocytes (Bonnet and Walsh, 2005; Kobayashi et al., 2005; †Significantly different from IL-1β alone (p<0.05). Cont: control, Loeser, 2006; Goldring et al., 2008). IL-1β is an inflammatory concentration unit is mM. cytokine that is produced by cells in articular tissues, including chondrocytes, and can increase expression of MMPs, thus stimulating the progression of osteoarthritis. IL-1β contributes IL-1β plus apigenin 50 μM, and IL-1β plus apigenin 100 μM, to the initiation and progression of destruction of articular car- respectively. tilage by suppressing synthesis of collagen and stimulating MMP expression (Aida et al., 2005; Kobayashi et al., 2005; Effect of apigenin on proteolytic activity of MMP-3 in Pantsulaia et al., 2010). Especially, it has been studied that rabbit articular chondrocytes MMP-3 plays a pivotal pathophysiological role in osteoarthri- To investigate the effect of apigenin on the enzyme activity tis by degrading components of the extracellular matrix, such of secreted MMP-3, which is known to degrade proteoglycans, as proteoglycans. Compared with the control group, patients 1 of the 2 major matrix components of cartilage, culture su- suffering from osteoarthritis in knee joints have shown more pernatants from rabbit articular chondrocytes were analyzed increased MMP-3 levels than MMP-1 levels (Garnero et al., for caseinolytic activity by casein zymography, after treatment 2000; Lijnen, 2002). with IL-1β for 24 h. As can be seen in Fig. 4, IL-1β increased As shown in Results, we have shown that apigenin inhib- the caseinolytic activity of MMP-3 in rabbit articular chondro- ited IL-1β-induced MMP-3 gene expression. However, another cytes, and this effect was inhibited by pretreatment with api- flavonoidal compound similar to apigenin, such as hesperidin genin. The relative signal intensity of each band of apigenin- or hesperetin, had no effect on MMP-3 gene expression (Fig. treated cultures were 100 ± 17%, 498 ± 27%, 283 ± 16%, 226 1A-1C). This suggests that apigenin suppresses the gene ex- ± 27%, 213 ± 21% and 170 ± 15% for control, 10 ng/mL of pression of MMP-3 at the transcriptional level. IL-1β alone, IL-1β plus apigenin 1 μM, IL-1β plus apigenin 10 In addition to MMP-3, it has also been reported that MMP-1 μM, IL-1β plus apigenin 50 μM, and IL-1β plus apigenin 100 and MMP-13 contribute to the destruction of cartilage in os- μM, respectively. teoarthritis. MMP-1 is a commonly detected metalloproteinase in synovial fluid from patients suffering from osteoarthritis Effect of apigenin on MMP-3 production in vivo (Freemont et al., 1997; Goupille et al., 1998; Kanyama et al., To investigate whether apigenin shows the potential effect 2000; Yoshihara et al., 2000; Neuhold et al., 2001; Jo et al., in vivo, we examined the effect of intraarticular injection of api- 2003; Little et al., 2009). Another degradative enzymes were genin into the knee joint of rats on IL-1β-stimulated production considered in this paper. ADAMTS-4 is a major aggrecanase of MMP-3 from articular cartilage tissues. As shown in Fig. 5, in cartilage of mouse and ADAMTS-5 has been known to be treatment with IL-1β (20 ng/30 μL) increased MMP-3 produc- important in cartilage matrix destruction during osteoarthritis tion in articular cartilage tissues. However, apigenin inhibited (Stanton et al., 2005; Echtermeyer et al., 2009). IL-1β-induced MMP-3 production, in vivo. The relative signal For that reason, we examined the effect of apigenin on ex- http://dx.doi.org/10.4062/biomolther.2015.217 168 Park et al. Apigenin and Osteoarthritis

pression of MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Du, H., Hao, J., Liu, F., Lu, J. and Yang, X. (2015) Apigenin attenuates acute myocardial infarction of rats via the inhibitions of matrix me- β As shown in Fig. 3, apigenin inhibited IL-1 -induced gene talloprotease-9 and inflammatory reactions. Int. J. Clin. Exp. Med. expression of MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, 8, 8854-8859. in rabbit chondrocytes. In other words, the chondroprotective Durigova, M., Roughley. P. J. and Mort, J. S. (2008) Mechanism of effects of apigenin are supported by its regulation of the gene proteoglycan aggregate degradation in cartilage stimulated with expression of diverse proteases involved in the destruction of oncostatin M. Osteoarthr. Cartil. 16, 98-104. articular cartilage in osteoarthritis. Echtermeyer, F., Bertrand, J., Dreier, R., Meinecke, I., Neugebauer, K., Fuerst, M., Lee, Y. J., Song, Y. W., Herzog, C., Theilmeier, G. and In order to test if apigenin influences the MMP-3 gene ex- Pap, T. (2009) Syndecan-4 regulates ADAMTS-5 activation and pression at the transcriptional level, we investigated whether cartilage breakdown in osteoarthritis. Nat. Med. 15, 1072-1076. apigenin affects IL-1β-induced secretion of MMP-3 proteins Freemont, A. J., Hampson, V., Tilman, R., Goupille, P., Taiwo, Y. and from rabbit articular chondrocytes. As shown in Fig. 4, stimu- Hoyland, J. A. (1997) Gene expression of matrix metalloprotein- lation with IL-1β increased the secretion of MMP-3 from chon- ases 1, 3, and 9 by chondrocytes in osteoarthritic human knee ar- drocytes, and this effect was suppressed by apigenin. This ticular cartilage is zone and grade specific. Ann. Rheum. Dis. 56, 542-549. result tells that apigenin can regulate the step of protein syn- Garnero, P., Rousseau, J. C. and Delmas. P. D. (2000) Molecular ba- thesis and secretion of MMP-3. sis and clinical use of biochemical markers of bone, cartilage, and As examining the effect of apigenin on the proteolytic activ- synovium in joint diseases. Arthritis Rheum. 43, 953-968. ity of secreted MMP-3, culture supernatants from rabbit articu- Goldring, M. B., Otero, M., Tsuchimochi, K., Ijiri, K. and Li, Y. (2008) lar chondrocytes stimulated with IL-1β for 24 h were analyzed Defining the roles of inflammatory and anabolic cytokines in carti- for caseinolytic activity by casein zymography. Fig. 4 shows lage metabolism. Ann. Rheum. Dis. 67, iii75-iii82. Goupille, P., Jayson, M. I., Valat, J. P. and Freemont, A. J. (1998) Ma- that the proteolytic activity of MMP-3 was increased when rab- trix metalloproteinases: the clue to intervertebral disc degenera- bit articular chondrocytes were stimulated with IL-1β, and this tion. Spine 23, 1612-1626. effect was suppressed by pretreatment with apigenin. From Hwang, Y. P., Oh, K. N., Yun, H. J. and Jeong. H. G. (2011) The flavo- this result, it is probable and possible that apigenin also af- noids apigenin and luteolin suppress ultraviolet A-induced matrix fect the proteolytic activity of overproduced and oversecreted metalloproteinase-1 expression via MAPKs and AP-1-dependent MMP-3 in osteoarthritic articular cartilage tissues. signaling in HaCaT cells. J. Dermatol. Sci. 61, 23-31. Jo, H., Park, J. S., Kim, E. M., Jung, M. Y., Lee, S. H., Seong, S. C., Moreover, we explored the effect of intraarticular injection of Park, S. C., Kim, H. J. and Lee, M. C. (2003) The in vitro effects apigenin into the knee joint of rats on IL-1β-stimulated produc- of dehydroepiandrosterone on human osteoarthritic chondrocytes. tion of MMP-3 in articular cartilage tissue. As can be seen in Osteoarthr. Cartil. 11, 585-594. Fig. 5, apigenin inhibited IL-1β-stimulated production of MMP- Kanyama, M., Kuboki, T., Kojima, S., Fujisawa, T., Hattori, T., Takiga- 3 in articular cartilage tissue. This result shows that, in addition wa, M. and Yamashita, A. (2000) Matrix metalloproteinases and to its in vitro effects, apigenin exerts chondroprotective effects tissue inhibitors of metalloproteinases in synovial fluids of patients with temporomandibular joint osteoarthritis. J. Orofac. Pain 14, 20- in vivo when administered via intraarticular injection. 30. In summary, our experiments show that the chondroprotec- Kobayashi, M., Squires, G. R., Mousa, A., Tanzer, M., Zukor, D. J., tive effects of apigenin are produced by its regulation of the Antoniou, J., Feige, U. and Poole, A. R. (2005) Role of interleukin-1 gene expression, secretion, and enzyme activity of MMP-3 in and tumor necrosis factor a in matrix degradation of human osteo- articular chondrocytes. 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